Promoting Recovery from Damage to the Central Nervous System

ABSTRACT

Methods, kits and compositions for improving a subject&#39;s recovery from CNS injury are disclosed. In certain aspects, a method may include administering to a subject cells and a neural stimulant. Recovery may be manifest by improvements in sensorimotor or cognitive abilities, e.g., improved limb movement and control or improved speech capability. In certain embodiments, subject methods can be used as part of a treatment for damage resulting from ischemia, hypoxia or trauma.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/642,277, filedAug. 18, 2000, which claims the benefit of U.S. Provisional ApplicationSer. No. 60/149,561, filed Aug. 18, 1999. The above-listed applicationsare hereby incorporated herein by reference in their entirety.

BACKGROUND

The central nervous system (CNS) is particularly vulnerable to insultsthat result in cell death or damage in part because cells of the CNShave a limited capacity for repair. As a result, disorders of the CNSoften result in debilitating and largely irreversible degradation of apatient's cognitive and sensorimotor functions. Conditions that resultin nerve cell death and damage range from degenerative disorders, suchas Alzheimer's disease, to ischemic episodes, such as stroke, to trauma.

Injury to the central nervous system (CNS) is an important cause ofdeath and disability worldwide. For example, stroke is the third leadingcause of death and disability in the U.S., with an estimated incidenceof 700,000 cases annually (Furie et al. (1998) “Cerebrovascular Disease”in The Atlas of Clinical Neurology, R.N. Rosenberg, Ed. CurrentMedicine: Philadelphia). Two-thirds of stroke patients survive the firstyear following stroke, for an average of seven years, leading to morethan 4.8 million stroke survivors currently in the U.S. Stroke costs theU.S. economy in excess of $30 billion per year in terms of medical costsand lost wages.

After several hours, little can be done to prevent the direct damage tothe CNS caused by CNS disorders. For example, stroke treatments musttypically be administered within six hours of onset. Depending on wherethe injury occurs in the brain, patients may be paralyzed on one side,may lose the ability to speak or see, and may have difficulty walking,among other symptoms. Gradual recovery of these functions is common,although recovery may be incomplete, and depends on the size andlocation of injury, among other factors.

Since damaged brain tissue does not regenerate, recovery must come fromthe remaining intact brain, which reorganizes itself, or rewires, inorder to compensate for some of the function lost by the damage. Indeed,studies in animals and humans provide ample evidence of suchreorganization of brain function following stroke. In particular,remaining neurons in both the damaged hemisphere and in the oppositeintact hemisphere grow new processes (both axons and dendrites) and formnew connections (synapses), which most likely contribute to recovery(Kawamata et al. (1997) Proc. Natl. Acad. Sci. USA, 94: 8179-8184; Joneset al. (1994) J. Neurosci., 14: 2140-2152; Stroemer et al. (1998)Stroke, 29: 2381-2395; Cramer et al. (1997) Stroke, 28: 2518-2527).

As an example, stroke treatment has focused on limiting the extent ofdamage within the first few hours. Stroke is generally due to a blockageof an artery leading to the brain, resulting in the death of brain cellssupplied by that artery. Current treatments for stroke have centered ontreatments to prevent arterial blockages (control of blood pressure,lipids, heart disease, etc.), and treatments to prevent brain damageonce the blockage has occurred. These latter treatments include“thrombolytic agents” (“clot busters” such as tPA) to break up arterialclots, and “neuroprotective agents,” designed to protect brain tissue atrisk for stroke. Such thrombolytic and neuroprotective agents must beadministered within hours after the onset of stroke in order to beeffective.

Currently there are only a few available methods of promoting recoveryin patients after cell death and injury has already occurred. Methods oftreating stroke after the initial phase of damage are mechanisticallydifferent from methods used in the first few hours. Treatments topromote recovery typically focus on encouraging neuronal growth andrewiring.

Direct application of neurotrophic growth factors to the brain canenhance spontaneous functional recovery occurring in animal models ofstroke (Kawamata et al. (1997) Proc. Natl. Acad. Sci. USA, 94:8179-8184; Kawamata et al. (1996) J. Cereb. Blood Flow Metab., 16:542-547; Kawamata et al. (1999) Exp. Neurol. 158: 89-96; Alps et al.,U.S. Pat. No. 5,733,871, Fisher et al. (1995) J. Cereb. Blood FlowMetab., 15: 953-959; Jiang et al. (1996) J. Neurol. Sci., 139: 173-179).For example, basic fibroblast growth factor (bFGF) is a protein thatsupports survival and axonal outgrowth from neurons. When bFGF isadministered starting a day or more after stroke, animals recover morequickly and to a greater extent on tests of sensorimotor function of theimpaired limbs (opposite to the side of the stroke). This recovery isnot due to a decrease in magnitude of the original brain damage.Instead, data suggests that this enhancement of recovery may be due toenhancement of new neuronal sprouting and synapse formation in theintact remaining brain tissue. Such remodeling appears to occur in boththe damaged and undamaged hemispheres. Other mechanisms of recovery mayinclude stimulation of endogenous neural stem cells within the brainthat then differentiate into neurons, replacing to some extent neuronslost by stroke.

Another potential approach to a treatment for stroke recovery includesthe use of neural stem cells. These are pluripotential cells alreadypresent in the developing and mature mammalian brain that, given theappropriate stimulation, can differentiate into brain neurons and/orglial cells. Several investigators have been successful in separatingand cloning out such neural stem cell lines from both the murine andhuman brain (Snyder et al. (1997) Proc. Natl. Acad. Sci. USA, 94:11663-11668; Gage et al. (1995) Proc. Natl. Acad. Sci. USA, 92:11879-11883; Kuhn et al. (1997) J. Neurosci., 17: 5820-5829; McKay etal., U.S. Pat. No. 5,270,191; Johe, K., U.S. Pat. No. 5,753,506;Carpenter, M., U.S. Pat. No. 5,968,829; Weiss et al., U.S. Pat. No.5,750,376). When such stem cells are reintroduced into the developing ormature brain, they can divide, migrate, grow processes, and assumeneural phenotypes, including the expression of neurotransmitters andgrowth factors normally elaborated by neurons. Thus, use of neural stemcells may be advantageous for stroke recovery in at least two ways: (1)by the stem cells partially repopulating dead areas and re-establishingneural connections lost by stroke, and (2) by secretion of importantneurotransmitters and growth factors required by the brain to rewireafter stroke. Efforts to promote recovery from brain injury in animalsusing neural stem cells have been described (Park et al. (1999) J.Neurotrauma 16: 675-687; Park et al. (1995) Soc. Neurosci. Abs. 21:2027; Stroemer et al. (1999) Soc. Neuroscience Abs. 25: 1310). Effortsusing a line of teratocarcinoma-derived cells have also been describedin animals (Borlongan et al. (1993) Int. J. Devl. Neuroscience 11:555-568) and humans (Kokaia et al. (1998) Eur. J. Neurosci., 10:2026-36).

Methods currently available for promoting recovery from CNS damage allowonly partial recovery of neurological functions. In patients sufferingfrom debilitating neurological deficits, incremental improvements infunction may have a significant effect on quality of life. Given thelarge number of affected patients and the limitations of currentmethods, there is an urgent need for additional and improved methods topromote recovery from damage to the nervous system. The modes oftreatment presented herein promote a greater degree of recovery from CNSdamage than is currently available with other known treatment methods.

SUMMARY

One aspect of the present application relates to methods for improving asubject's recovery from CNS injury or damage. In one aspect, theinvention comprises administering to a subject cells, preferably stemcells, and a neural stimulant in sufficient amounts to improve thesubject's sensorimotor or cognitive abilities, e.g., improved limbmovement and control or improved speech capability.

In another aspect, the invention provides kits for the treatment of CNSdamage. In certain embodiments, kits of the invention comprise stemcells and a neural stimulant. In other embodiments, the kits of theinvention comprise a neural stimulant and a device for obtaining a stemcell-containing sample from a subject. In preferred embodiments, thekits comprise a polypeptide growth factor, and more preferably apolypeptide at least 30% identical but most preferably at least 40%,50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% identical to one of thepolypeptides of SEQ ID NOS: 1-3.

In a further aspect, the invention provides pharmaceutical preparationscomprising stem cells, a neural stimulant and one or morepharmaceutically acceptable reagents.

In preferred embodiments, stem cells for use in the invention are cellscapable of giving rise to brain cells, e.g. neurons, oligodendroglia orastroglia. In particularly preferred embodiments, stem cells are neuralstem cells, hematopoietic stem cells, teratocarcinoma-derived cells orembryonic stem cells. In other preferred embodiments, stem cells areobtained from the subject, and optionally cultured or enriched in vitroprior to administration.

In other embodiments, stem cells of the invention may be induced toproliferate in vitro by transfection with a gene encoding one or moreproliferation promoting factors, such as vmyc, SV40 T antigen, polyomavirus large T antigen, the neu oncogene or the ras oncogene. Inpreferred embodiments, the gene is strongly expressed in vitro,promoting proliferation, and poorly expressed after the cell has enteredthe central nervous system, such that the cell does not proliferaterapidly in vivo.

In a further embodiment, the neural stimulant is a polypeptide growthfactor. Preferred polypeptide growth factors comprise a polypeptide thatis chosen from among the following polypeptide families: fibroblastgrowth factor family members, neurotrophin family members, insulin-likegrowth factor family, ciliary neurotrophic growth factor family members;EGF family members, TGFβ family members, leukemia inhibitory factor(LIF); oncostatin M, interleukin-11; interleukin-6; members of theplatelet-derived growth factor family, and VEGF family members. It iscontemplated that, in certain embodiments, combinations of factors maybe used. Preferred polypeptides comprise a polypeptide with a sequencethat is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or100% percent identical to an amino acid sequence shown in any of SEQ IDNOS: 1-3.

In still other embodiments, the neural stimulant is a modulator ofneurotransmitter activity (e.g. an agonist or antagonist). In preferredembodiments, the neural stimulant is an antidepressant, such as Prozac,an amphetamine, Ritalin, a tricyclic antidepressant such as ELAVIL, orcombinations thereof. In another embodiment, the neural stimulant is apromoter of neuronal differentiation such as retinoic acid. In yetanother embodiment, the neural stimulant is a so-called guidancemolecule such as a netrin, a semaphorin, a neuropilin or an ephrin. Inyet an additional embodiment, the neural stimulant may be transcranialmagnetic stimulation.

In another aspect, the invention comprises conjoint administration ofcells with a bioactive compound that is not a neural stimulant.Preferred bioactive compounds include immunosuppressants such asimmunophilins (e.g. cyclosporin, FK506, and thalidomide) andantibiotics, such as tetracycline.

A range of techniques for administering the cells and neural stimulantsof the invention are contemplated. Cells and neural stimulants do notneed to be administered in the same way or at the same time, but theyare preferably administered such that their effects overlap. Inpreferred embodiments, administration is carried out at least 6,10,12 or24 hours after the injury has occurred.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a graph that depicts the results of forelimb placing tests ina rat stroke model. Rats were treated with bFGF alone, stem cells alone,bFGF and stem cells, or control vehicle only.

FIG. 1B is a graph that illustrates the results of hindlimb placingtests in a rat stroke model.

FIG. 1C is a graph that illustrates the results of bodyswing tests in arat stroke model.

FIG. 1D is a graph showing the results of spontaneous limb use tests ina rat stroke model.

FIG. 2A is a graph that depicts the results of forelimb placing tests ina rat stroke model. Rats were treated with bFGF alone, stem cells alone,bFGF and stem cells, or control vehicle only.

FIG. 2B is a graph that illustrates the results of hindlimb placingtests in a rat stroke model.

FIG. 2C is a graph that illustrates the results of bodyswing tests in arat stroke model.

FIG. 2D is a graph showing the results of spontaneous limb use tests ina rat stroke model.

FIG. 3 is a graph depicting the results of paw reaching tests in a ratstroke model.

FIG. 4 presents amino acid sequences for variants of bFGF (SEQ IDNOS:1-3).

DETAILED DESCRIPTION

4.1 Definitions:

The term “brain cells” as used herein refers to cells comprising thebrain, including neurons, astroglia, oligodendroglia, and microglia.Many specific cell types belong to each category. For example, neuronsinclude dopaminergic, cholinergic and glutaminergic neurons, to nameonly a few.

“Bioactive compounds” is intended to include compounds with a desirableeffect when used within the context of the invention. Bioactivecompounds include many neural stimulants (see below) as well as manycompounds that are not considered neural stimulants but that also havedesirable effects. For example, immunosuppressants such as theimmunophilins (e.g. FK506), can exhibit the dual action of preventingrejection of the transplanted cells and providing a neuroprotectiveactivity (Bavetta et al. (1999) Exp. Neurol. 158: 382-393). Antibiotics,and particularly tetracyclines, can suppress possible infections andalso have beneficial effects on neural cells (Yrjanheikki et al. (1998)PNAS 95: 15769-74).

“Cell culture” refers generically to any composition of cells whetheractively growing, differentiating, or static. Cell cultures can take ona variety of formats. For instance, a “suspension culture” refers to aculture in which cells are suspended in a suitable medium. A “continuousflow culture” refers to the cultivation of cells in a continuous flow offresh medium to maintain cell growth or viability. “Continuousexpansion” is a method of growing cells by continuous flow culture.

The “central nervous system” (CNS) as used herein, refers to anycomponent of the central nervous system including the brain and spinalcord, the cells and extracellular materials and fluids.

“Conjoint administration” is used herein in reference to theadministration of cells and a neural stimulant or bioactive compound tosubjects. The term “conjoint administration” is not meant to indicatethat the cells and the neural stimulant must be administered at the sametime. The components of the conjoint administration may be delivered atdifferent times, at different time intervals and by different means. Theadministrations should, however, overlap in therapeutic effects.

The term “culture medium” is recognized in the art, and refers generallyto any substance or preparation used for the cultivation of livingcells.

The term “developmental regulator” is used herein to refer to moleculesthat modulate development in brain cells or stem cells with the capacityto become brain cells.

By “focal cerebral ischemia” as used herein in reference to the centralnervous system, is meant the condition that results from the blockage ofa single artery that supplies blood to the brain or spinal cord,resulting in the death of cellular elements in the territory supplied bythat artery.

“Global cerebral ischemia” is the diminution of blood flow to the entirebrain, often caused by cardiac arrest or hypotension, for example. Inglobal cerebral ischemia, cells that are particularly vulnerable toischemia tend to die or become injured, resulting in patches of damagedistributed around the brain. This differs from the type of damage thatoccurs in focal cerebral ischemia.

“Guidance molecules” are a class of proteins, normally found in theextracellular matrix, that function to guide cells or cellular processes(axons) to locations required for proper functioning. Examples are thesemaphorins, the netrins, the neuropilins, and the ephrins. (Perris etal. (2000) Mech. Dev. 95: 3-21; Wilkinson (2000) Int. Rev. Cytol. 196:177-244; Van Vactor et al. (1999) Curr. Biol. 9: R201-4).

“Hematopoietic stem cells” (HSCs) as used herein are stem cells that cangive rise to cells of at least one of the major hematopoietic lineagesin addition to producing daughter cells of equivalent potential. Threemajor lineages of blood cells include the lymphoid lineage, e.g. B-cellsand T-cells, the myeloid lineage, e.g. monocytes, granulocytes andmegakaryocytes, and the erythroid lineage, e.g. red blood cells. CertainHSCs are capable of giving rise to many other cell types including braincells. “Multipotent” or “pluripotent” HSCs are HSCs that can give riseto at least three of the major hematopoietic lineages.

“Homology” and “identity” each refer to sequence similarity between twopolypeptide sequences, with identity being a more strict comparison.Homology and identity can each be determined by comparing a position ineach sequence which may be aligned for purposes of comparison. When aposition in the compared sequence is occupied by the same amino acidresidue, then the polypeptides can be referred to as identical at thatposition; when the equivalent site is occupied by the same amino acid(e.g., identical) or a similar amino acid (e.g., similar in stericand/or electronic nature), then the molecules can be referred to ashomologous at that position. A percentage of homology or identitybetween sequences is a function of the number of matching or homologouspositions shared by the sequences. An “unrelated” or “non-homologous”sequence shares less than 40 percent identity, though preferably lessthan 25 percent identity, with the polypeptide sequence of a bioactivepolypeptide of the present invention.

The term “ischemic episode” is used to mean any circumstance thatresults in a deficient supply of blood to a tissue. Cerebral ischemicepisodes result from a deficiency in the blood supply to the brain. Thespinal cord, which is also a part of the central nervous system, isequally susceptible to ischemia resulting from diminished blood flow. Anischemic episode may be caused by a constriction or obstruction of ablood vessel, as occurs in the case of a thrombus or embolus.Alternatively, the ischemic episode can result from any form ofcompromised cardiac function, including cardiac arrest.

The term “neural stimulant” refers to a treatment that affects neuralfunction or activity. Such treatments are typically polypeptide growthfactors, for example neurotrophins or fibroblast growth factors. Suchtreatments also include guidance molecules and non-polypeptide moleculesthat are active in the brain, such as neurotransmitters,neurotransmitter antagonists or agonists, and developmental regulators.“Neural stimulants” may also be agents that affect the same signalingtransduction pathways as those affected by the above listed agents. Forexample, a chemical that activates bFGF receptor signaling could be usedas a neural stimulant. A “neural stimulant” can also include otherchemical or electromagnetic treatments that alter the production ofmolecules that affect neural function or activity (e.g. transcranialmagnetic stimulation).

“Neural stem cell” (NSC) is used to describe a cell derived from tissueof the central nervous system, or the developing nervous system, thatcan give rise to at least one of the following fundamental neurallineages: neurons, oligodendroglia and astroglia. Additionally, a neuralstem cell must also be able to give rise to new NSCs with similarpotential. “Multipotent” or “pluripotent” NSCs are NSCs that are capableof giving rise to all of the above neural lineages as well as cells ofequivalent developmental potential.

“Neuronal function” is used to refer generally to all the functions ofthe nervous system, e.g. sensorimotor function and cognitive function.

“Neuroepithelial stem cells” are stem cell populations isolated fromfetal neuroepithelial tissue. Such cells may be considered a subset ofneural stem cells, as used herein. “Neuroepithelial cells” tend to bemultipotent.

“Neurotransmitters” are small molecules released from an axon for actionwithin a synapse. Exemplary neurotransmitters include catecholamines(e.g. epinephrine, norepinephrine and dopamine), serotonin,acetylcholine, glutamate and GABA.

A “patient” or “subject” to be treated by the subject method is amammal, including a human.

As used herein, both “protein” and “polypeptide” mean any chain of aminoacid residues, regardless of length or post-translational modification(e.g., glycosylation or phosphorylation). A “bioactive polypeptide”, asused herein is a polypeptide that has activity as a neural stimulant.Examples are polypeptide growth factors and guidance molecules.“Bioactive polypeptides” also include active fragments and analogues ofthe bioactive polypeptides, which possess one or more the biologicalfunctions of those factors.

“Polypeptide growth factors” are generally secreted polypeptides, oractive fragments thereof, that stimulate cell growth or growth of cellprocesses (e.g. axons, dendrites etc.) in at least on cell type.

“Active fragment” as used in reference to bioactive polypeptides,indicates any portion of a polypeptide that has at least one activity ofthe full-length polypeptide. Many polypeptides have several differentactivities and it may be desirable to use an active fragment that hasonly one or a subset of these activities. The active fragment willproduce at least 20%, preferably at least 50%, more preferably at least70%, and most preferably at least 90% (including up to 100%) of theactivity of the full-length polypeptide. An example is bFGF, which canbe polymorphic, with observed molecular weights of 17.8, 22.5, 23.1, and24.2 kDa; all of these forms are biologically active and can be used inthe invention.

The terms “recombinant protein”, “heterologous protein” and “exogenousprotein” are used interchangeably throughout the specification and referto a polypeptide which is produced by recombinant DNA techniques,wherein generally, DNA encoding the polypeptide is inserted into asuitable expression construct which is in turn used to transform a hostcell to produce the heterologous protein. That is, the polypeptide isexpressed from a heterologous nucleic acid.

A “stroke” is a sudden loss of function caused by an abnormality in theblood supply to the brain. Stroke presents with different levels ofseverity ranging from “transient ischemic attack” or “TIA” (no permanentdisability), to “partial nonprogressing stroke” (persistent but nocalamitous damage), to “complete stroke” (permanent, calamitousneurological deficit). Ischemia (diminished or stopped blood flow) andinfarction (cell damage and death within the zone of ischemia) are thepathologic processes in stroke that lead to neurologic deficits.“Ischemic stroke” is caused by an obstruction of blood vessels supplyingthe brain. The primary subcategories of ischemic stroke are thromboticstroke, embolic stroke and lacunar infarctions. “Hemorrhagic stroke” iscaused by the rupture of blood vessels supplying the brain. The primarysubcategories of hemorrhagic stroke are subarachnoid hemorrhage (SAH)and intracerebral hemorrhage (ICH).

A “therapeutically effective amount” of, e.g. cells or neural stimulant,with respect to the subject method, refers to an amount of thetherapeutic (in a preparation) which when applied as part of a desireddosage regimen causes an improvement in neuronal function according toclinically acceptable standards.

“Transcranial magnetic stimulation” (TMS) is a method for thestimulation of neurons by briefly generating magnetic fields withtypical field strengths between 2 and 4T using coils close to the head(currents in TMS coils can be has high as 8000A). TMS often involvespulses of stimulation with varying pulse and delay times. TMS is knownto upregulate monoamines in the brain.

4.2 Overview:

The present invention is based in part on the surprising finding thatthe conjoint administration of cells and neural stimulants promotesgreater recovery from CNS damage than either treatment alone. In certainaspects, the invention provides improved methods, compositions and kitsfor stimulating recovery of damaged brain tissue, whether damage islocalized or global. In preferred embodiments, the invention pertains torecovery from ischemia, hypoxia and trauma. In certain aspects, themethods of the invention comprise the conjoint administration of stemcells and a neural stimulant, e.g. a polypeptide growth factor or othermolecule. The conjoint treatment gives a greater degree of recovery thanhas been possible with either treatment alone. The promise of thisapproach was recently illustrated in a study wherein the polypeptidegrowth factor BDNF was administered conjointly with bone marrow cells toimprove recovery in a rat stroke model (Chen et al., 2000,Neuropharmacology 39: 711-716). The debilitating effects of CNS damageare such that even incremental improvements in recovery can lead tomajor improvements in a patient's quality of life.

The subject method has wide applicability to the treatment of CNSdamage. In this regard, the subject method is useful for, but notlimited to, treatment of injury to the brain and spinal cord due toischemias, hypoxia, traumas, neurodegenerative diseases, infectiousdiseases, cancers, autoimmune diseases and metabolic disorders. Examplesof disorders include stroke, head trauma, spinal trauma, hypotension,arrested breathing, cardiac arrest, Reye's syndrome, cerebralthrombosis, embolism, cerebral hemorrhage, brain tumors,encephalomyelitis, hydroencephalitis, operative and postoperative braininjury, Alzheimer's disease, Huntington's disease, Creutzfeld-Jakobdisease, Parkinson's disease, multiple sclerosis and amyotrophic lateralsclerosis.

Thrombus, embolus, and systemic hypotension are the most common causesof cerebral ischemic episodes. Other causes of cerebral ischemia includehypertension, hypertensive cerebral vascular disease, rupture of ananeurysm, an angioma, blood dyscrasias, cardiac failure, cardiac arrest,cardiogenic shock, septic shock, head trauma, spinal cord trauma,seizure, bleeding from a tumor, or other blood loss. With respect totrauma, trauma can involve a tissue insult such as an abrasion,incision, contusion, puncture, compression, etc., such as can arise fromtraumatic contact of a foreign object with any locus of or appurtenantto the head, neck, or vertebral column. Other forms of traumatic injurycan arise from constriction or compression of the CNS tissue by aninappropriate accumulation of fluid (for example, a blockade ordysfunction of normal cerebrospinal fluid or vitreous humor fluidproduction, turnover, or volume regulation, or a subdural orintracranial hematoma or edema). Similarly, traumatic constriction orcompression can arise from the presence of a mass of abnormal tissue,such as a metastatic or primary tumor.

In some cases the damage caused by the above disorders is primarilylocated in a single region of the brain, e.g. focal ischemia, certaintraumas and Parkinson's disease. In other cases, damage can be morewidespread or distributed across disparate regions of the brain, e.g.hypoxia and global ischemia, and Creutzfeld-Jakob disease. Becausecertain cells of the invention are known to migrate freely throughoutthe brain, and because growth factors can be provided so as to begenerally available to all brain tissues, it is anticipated that themethods and compositions of the invention will be useful in promotingrecovery from both global and focal brain damage.

In a general outline, a treatment protocol of the invention involvesadministering a neural stimulant and stem cells to a patient that hassuffered CNS damage. In preferred embodiments, CNS damage was caused byischemia, hypoxia or trauma. Treatment may include obtaining cells fromthe patient, optionally enriching for therapeutically useful cells, andadministering the cells to the patient. In this way, the patient is notsubjected to any foreign cells, which offers the advantage of avoidingimmune responses to the cells.

The treatment regimen according to the invention is carried out, interms of administration mode, timing of the administration, and dosage,so that the functional recovery of the patient from the adverseconsequences of the central nervous system injury is improved; forexample, the patient's motor skills (e.g., posture, balance, grasp, orgait), cognitive skills, speech, and/or sensory perceptions (includingvisual ability, taste, olfaction, and proprioception) may improve asresult of treatment according to the invention.

While not wishing to be limited to a particular mechanism of action, itis believed that the methods of the invention promote recovery from CNSdamage by stimulation of neuronal sprouting and new synapse formation.In cases of stroke, essentially all current treatments focus on infarctreduction and prevention of damage. Therefore, the present inventionrelates to unconventional and novel methods of treating CNS damage.

4.3 Neural Stimulants and Other Bioactive Factors:

Neural stimulants of the invention include treatments, chemical orotherwise, that affect neural function or activity. Such treatments aretypically bioactive polypeptides, but non-polypeptide molecules andphysical treatments such as transcranial magnetic stimulation are alsocontemplated.

In one set of preferred embodiments, the neural stimulant is apolypeptide growth factor. The polypeptide growth factor can beadministered in a pharmaceutically acceptable carrier, and may also beadministered mixed or unmixed with cells. The polypeptide growth factorcan be a member of the fibroblast growth factor (FGF) family; theneurotrophin family; the insulin-like growth factor (IGF) family; theciliary neurotrophic growth factor (CNTF) family; the EGF family; theTGF-beta family; the PDGF family; the VEGF family; the leukemiainhibitory factor (LIF) family; an interleukin (e.g. IL-11, IL-6, IL-1);or an oncostatin (e.g. oncostatin M). Characteristics and exemplarymembers of each of these families are given below and in Table 2. Inpreferred embodiments the polypeptide factor is a human polypeptidefactor.

The FGF family contains at least 15 distinct factors that are highlyconserved across mammalian species, although individual family memberscan be highly divergent from each other (generally 30-70% sequenceidentity). FGFs are secreted proteins that share a basic tertiarystructure composed of 12 beta-strands in a beta-trefoil fold. Mostfamily members have mitogenic effects on various cell types and alsobind heparin. Exemplary members of the FGF family include: basic FGF(bFGF, FGF-2), acid FGF (aFGF, FGF-1), FGF-3 (int-2), FGF-4 (hst/kFGF),FGF-5, FGF-6, FGF-7 (KGF), FGF-8 (AIGF), and FGF-9 (GAF). (Stauber etal. (2000) PNAS 97: 49-54; Wong et al. (1998) J. Biol. Chem. 273:18617-18622; Szebenyi et al. (1999) Int. Rev. Cytol. 185: 45-106).

The neurotrophin family includes several related, secreted factors thatexert their effects primarily on the nervous system. Neurotrophins aregenerally produced as precursor proteins that are highly processed togive the mature forms. Mature neurotrophins carry a set of six cysteinesthat engage in disulfide bonding in the order 1-4 (i.e. the first andfourth cysteines form a disulfide bond), 2-5 and 3-6. Typically,neurotrophin family members have a surface composed of 3 antiparallelbeta-strands, and dimerization occurs along this surface. Exemplarymembers of the neurotrophin family are: nerve growth factor (NGF),brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3),neurotrophin 4/5 (NT4/5) and neurotrophin 6. (Lewin et al. (1996) Annu.Rev. Neuroscience 19:289-317).

The insulin-like growth factor family includes secreted proteins with asequence and structure similar to that of insulin and a molecular weighttypically in the range of 5-10 kDa. These factors can be found in thebloodstream, usually associated with one of six IGF binding proteins.Exemplary members of the family include IGF-1 and IGF-2. IGF-1 and -2are known to promote recovery from various insults to the CNS.(Daughaday et al. (1989) Endocr. Rev. 10: 68-91; Rajaram et al. (1997)Endocr. Rev. 18: 801-831; Jones et al. (1995) Endocr. Rev. 16: 3-34).

The epidermal growth factor family is a large family of related secretedfactors. Members of the EGF family share at least 30% sequence homologyand a set of six conserved cysteine residues in the C-terminal end ofthe protein. Most such proteins also contain an EGF-like domain, whichis a particularly well-characterized domain that is also present in manynon-EGF family member proteins. EGF family members are normallyprocessed from larger precursors. Exemplary members of the EGF familyinclude EGF, TGF-alpha, HB-EGF (heparin-binding EGF), amphiregulin,betacellulin, vaccinia growth factor and neu differentiation factor.(Aviezer et al. (1994) 91: 12173-12177; Higashyama et al. (1992) J.Biol. Chem. 267: 6205-6212; Pelles et al. (1992) Cell 69:205-216).

The TGF-beta superfamily is an important class of molecules involved incell-cell signaling and development in a wide range of organisms andcell types. Members of the family are initially synthesized as largerprecursor molecules with an amino-terminal signal sequence and apro-domain of varying size (Kingsley, D. M. (1994) Genes Dev.8:133-146). The precursor is then cleaved to release a maturecarboxy-terminal segment of 110-140 amino acids. The active signalingmoiety is comprised of hetero- or homodimers of the carboxy-terminalsegment (Massague, J. (1990) Annu. Rev. Cell Biol. 6:597-641). Theactive form of the molecule then interacts with its receptor, which forthis family of molecules is composed of two distantly relatedtransmembrane serine/threonine kinases called type I and type IIreceptors (Massague, J. et al. (1992) Cell 69:1067-1070; Miyazono, K. A.et al. EMBO J. 10:1091-1101). TGF-beta binds directly to the type IIreceptor, which then recruits the type I receptor and modifies it byphosphorylation. The type I receptor then transduces the signal todownstream components (Wrana et al, (1994) Nature 370:341-347). Ingeneral, members of the TGF-beta superfamily have a set of nine highlyconserved cysteine residues that are involved in disulfide bonding bothwithin and between monomers of the mature, dimerized signaling protein(Griffith et al. (1996) PNAS 93: 878-883; Luo et al. (1995) PNAS 92:11761-11765; Schlunegger et al. (1993) J. Mol. Biol. 231: 445-58; Daopinet al. (1993) Proteins 17: 176-92; Murray-Rust et al. (1993) Structure15: 153-9; Archer et al. (1993) Biochemistry 32: 1164-71; Daopin et al.(1992) Science 257: 369-373; Schlunegger et al. (1992) Nature 358:430-434; Hinck et al. (1996) Biochemistry 35: 8517-34; Mittl et al.(1996) Protein Sci. 5:1261-71).

The transforming growth factor beta family is a very large family ofproteins including the TGF-beta subfamily, the bone morphogenesisprotein (BMP) subfamily, the activin subfamily, and others. Exemplarymembers of the TGF-beta subfamily include TGF-beta-1, -2, -3, -4 and -5.Exemplary members of the BMP subfamily include osteogenic protein 1(OP-1, BMP-7) and BMP-9. (Ren et al. (2000) Neuropharmacology 39:860-865; Lopez-Coviella et al. (2000) Science 289: 313-316; Withers etal. (2000) Eur. J. Neurosci. 12: 106-116).

The vascular endothelial growth factor (VEGF) family is a group ofsecreted proteins that act as potent mitogens in embryonic and somaticangiogenesis. VEGF proteins, including VEGF itself, bind to cell surfacereceptors of the kinase domain receptor family (KDR) and fms-liketyrosine kinase group (Flt receptors). VEGF proteins form a homodimerwith a cystine knot structure. Platelet-derived growth factor (PDGF)shares only limited sequence similarity with VEGF (19%) but hassubstantial structural similarity. PDGF and related family members arealso cystine knot proteins and bind to their receptors in a similarmanner. (Lobsiger et al. (2000) Glia 30: 290-300; Sun et al. (1995)Annu. Rev. Biophys. Biomolec. Struct. 24: 269-291; Muller et al. (1997)Structure 5: 1325-1338; Jiang et al. (2000) EMBO J. 19: 3192-3203;Muller et al. (1997) PNAS 94: 7192-7197).

Interleukins are secreted polypeptide factors that mediate signalingbetween immune cells. Many interleukins are known to have effects on thebrain, particularly IL-1# and #, IL-6 and IL-11. (Van Wagoner et al.(1999) J. Neuroimmunol. 100:124-139; Ling et al. (1998) Exp. Neurol.149: 411-23; Mehler et al. (1993) Nature 362: 62-5). Intriguingly, IL-6and IL-11 both act in part through a receptor protein gpl 30 that actsas a receptor for ciliary neurotrophic factor (CNTF), leukemiainhibitory factor (LIF) and oncostatin M. Thus all these factors mayhave similar roles in modulating neuronal function and development.(Benigni et al. (1995) Mol. Med. 1: 568-75; Benigni et al. (1996) Blood87: 1851-4; Murphy et al. (1997) Prog. Neurobiol. 52: 355-78).

Table 1: Polypeptide Growth Factors TABLE 1 Polypeptide Growth FactorsExemplary Family subfamilies Exemplary Members FGF bFGF, aFGF, FGF-3,FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9 Neurotrophins NGF, BDNF, NT3,NT4/5, NT-6 IGF IGF-1, IGF-2 EGF EGF, TGF-alpha, HB-EGF, amphiregulin,betacellulin, vaccinia growth factor and neu TGF-beta TGF-betaTGF-beta-1, -2, -3, -4 and -5 BMP OP-1, BMP-9 Activin InhibinβA,InhibinβB and InhibinβC VEGF VEGF PDGF PDGF LIF LIF CNTF CNTFInterleukins IL-1α, IL-1β, IL-6, IL-11 Oncostatins Oncostatin M

Furthermore, the nomenclature in the field of polypeptide factors iscomplex, primarily because many factors have been isolated independentlyby different groups of researchers and, historically, named for the typeof tissue that was used as an assay in the process of purifying thefactor. Basic FGF has been referred to in scientific publications by anumber of different names, and has multiple family members. Theseinclude leukemic growth factor, macrophage growth factor, embryonickidney-derived angiogenesis factor 2, prostatic growth factor,astroglial growth factor 2, endothelial growth factor, chondrosarcomagrowth factor, cartilage-derived growth factor 1, eye-derived growthfactor 1, heparin-binding growth factors class 11, myogenic growthfactor, human placenta purified factor, uterine-derived growth factor,embryonic carcinoma-derived growth factor, human pituitary growthfactor, adipocyte growth factor, prostatic osteoblastic factor, andmammary tumor-derived growth factor. Thus, any factor referred to by oneof the aforementioned names is considered within the scope of theinvention. Furthermore, effort has been made to use commonly acceptednames for factors, and any factor listed here is considered within thescope of the invention regardless of whether it is known to others by adifferent name.

The invention can also employ bioactive analogues of the aforementionedgrowth factors, which possess one or more of the biological functions ofthose factors. An example is bFGF, which can be polymorphic, withobserved molecular weights of 17.8, 22.5, 23.1, and 24.2 kDa; all ofthese forms are biologically active and can be used in the invention. Itis possible to identify bioactive analogues of the aforementionedfactors. Such analogues, when designed to retain at least one activityof a naturally occurring form of the polypeptide, are consideredfunctional equivalents. Bioactive analogues may also include moleculesthat are not polypeptides but nonetheless mimic activities of apolypeptide growth factor. Bioactive analogues may also haveadvantageous properties, such as enhanced efficacy or more desirablestability properties (e.g., ex vivo shelf life and resistance toproteolytic degradation in vivo). For example, the analogue may berendered either more stable or less stable to proteolytic degradation orother processes which result in destruction of, or otherwiseinactivation of, the factor. A short half-life can give rise to moretransient biological effects can therefore allow tighter control ofprotein levels within or around a particular tissue. A longer half-lifecan increase the potency of the factor.

In certain embodiments, bioactive polypeptides of the invention comprisea polypeptide with an amino acid sequence that is at least 30% identicalto the bFGF sequence set forth in one of SEQ ID NOS: 1-3. In preferredvariations, such bioactive polypeptides comprise a polypeptide with anamino acid sequence that is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%,98%, 99% or 100% identical to one of SEQ ID NOS: 1-3.

Methods for generating such bioactive analogues are well known in theart. In general, variations of a polypeptide factor can be generated byintroducing changes into a nucleic acid sequence encoding the factor.The altered nucleic acid can then be expressed to produce alteredpolypeptides, and the polypeptides can be assayed for variousproperties. Changes in nucleic acid sequences can be made individuallyto introduce particular, desired changes. Alternatively, libraries ofsemi-randomly generated variants may be produced and screened foractivity.

There are many ways by which a library of potential bioactive analogscan be generated. In an illustrative embodiment, the amino acidsequences for a population of bFGF homologs or other related proteinsare aligned, preferably to promote the highest homology possible. Such apopulation of variants can include, for example, bFGF homologs from oneor more species, e.g. murine and chicken, or bFGF homologs from the samespecies but which differ due to mutation. Amino acids which appear ateach position of the aligned sequences are selected to create adegenerate set of combinatorial sequences. In a preferred embodiment,the variegated library of bFGF variants is generated by combinatorialmutagenesis at the nucleic acid level, and is encoded by a variegatedgene library. For instance, a mixture of synthetic oligonucleotides canbe enzymatically ligated into gene sequences such that the degenerateset of potential bFGF sequences are expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins (e.g.for phage display) containing the set of bFGF sequences therein.

Chemical synthesis of a degenerate gene sequence can be carried out inan automatic DNA synthesizer, and the synthetic genes then ligated intoan appropriate expression vector. The purpose of a degenerate set ofgenes is to provide, in one mixture, all of the sequences encoding thedesired bioactive analogs. The synthesis of degenerate oligonucleotidesis well known in the art (see for example, Narang, SA (1983) Tetrahedron39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rd Cleveland Sympos.Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura etal. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477. Such techniqueshave been employed in the directed evolution of other proteins (see, forexample, Scott et al. (1990) Science 249:386-390; Roberts et al. (1992)PNAS 89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla etal. (1990) PNAS 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409,5,198,346, and 5,096,815).

Alternatives to the above combinatorial mutagenesis also exist. Forexample, bFGF analogs can be generated, for example, alanine scanningmutagenesis and the like (Ruf et al. (1994) Biochemistry 33:1565-1572;Wang et al. (1994) J. Biol. Chem. 269:3095-3099; Balint et al. (1993)Gene 137:109-118; Grodberg et al. (1993) Eur. J. Biochem. 218:597-601;Nagashima et al. (1993) J. Biol. Chem. 268:2888-2892; Lowman et al.(1991) Biochemistry 30:10832-10838; and Cunningham et al. (1989) Science244:1081-1085), by linker scanning mutagenesis (Gustin et al. (1993)Virology 193:653-660; Brown et al. (1992) Mol. Cell Biol. 12:2644-2652;McKnight et al. (1982) Science 232:316); by saturation mutagenesis(Meyers et al. (1986) Science 232:613); by PCR mutagenesis (Leung et al.(1989) Method Cell Mol Biol 1:11-19); or by random mutagenesis (Milleret al. (1992) A Short Course in Bacterial Genetics, CSHL Press, ColdSpring Harbor, N.Y.; and Greener et al. (1994) Strategies in Mol Biol7:32-34).

The above methods may be generalized to other polypeptide factors inaddition to bFGF.

Having generated one or more variants of a bioactive factor, variousmethods may be used to identify variants with the desired properties.Whether one or more changes in the amino acid sequence of a peptideresults in a bioactive analog can be readily determined by assessing theability of the variant peptide to produce a response in cells in afashion similar to the wild-type peptide or competitively inhibit such aresponse. In addition, the ability of such a polypeptide to bind to itsreceptor can also be determined. For example, bFGF normally binds to thereceptors FGFR1 and FGFR2. This binding is also stimulated by heparinbinding. These properties could be checked to verify that a bFGF variantis active.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries, and for screening cDNA librariesfor gene products having a certain property. The most widely usedtechniques for screening large gene libraries typically comprise cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates relatively easy isolation of the vector encodingthe gene whose product was detected. Each of the illustrative assaysdescribed below are amenable to high through-put analysis as necessaryto screen large numbers of variant sequences created by combinatorialmutagenesis techniques.

In one possible screening assay, the gene library is expressed as afusion protein on the surface of a viral particle. For instance, in thefilamentous phage system, foreign peptide sequences can be expressed onthe surface of infectious phage. These phage can be applied to affinitymatrices at very high concentrations, allowing screening of a largenumber of phage simultaneously. If a particular phage is recovered froman affinity matrix in low yield, the phage can be amplified by anotherround of infection in a suitable host, such as E. coli. The group ofalmost identical E. coli filamentous phages M13, fd, and F1 are mostoften used in phage display libraries, as either of the phage gIII orgVIII coat proteins can be used to generate fusion proteins withoutdisrupting the ultimate packaging of the viral particle (Ladner et al.PCT publication WO 90/02909; Garrard et al., PCT publication WO92/09690; Marks et al. (1992) J. Biol. Chem. 267:16007-16010; Griffithset al. (1993) EMBO J 12:725-734; Clackson et al. (1991) Nature352:624-628; and Barbas et al. (1992) PNAS 89:4457-4461).

In another embodiment, the combinatorial library is designed to beextracellularly presented (e.g. as it occurs naturally) or optionally,secreted (e.g. the polypeptides of the library all include a signalsequence). The library can be transfected into a eukaryotic cell thatcan be co-cultured with cells which express a functional receptor forthe desired bioactive fragment. For example, one might use cellsexpressing a bFGF receptor to identify bioactive variants of bFGF.Bioactive analogs secreted by the cells expressing the combinatoriallibrary will diffuse to neighboring receptor positive cells and induce aphenotypic change. Phenotypic changes may be detected using, forexample, antibodies directed to epitopes that are either created ordestroyed in response to factor treatment.

Each of these analogs can subsequently be screened for furtherbiological activities. For example, receptor-binding analogs isolatedfrom the combinatorial library can be tested for their effect oncellular proliferation relative to the wild-type form of the protein.Alternatively, one could screen the analogs for stability in vitro or invivo. The activity of such analogs can also be assessed in animalmodels. For example, the ability of an analog to improve neural functionin a rat stroke model could be assessed to verify that an analog has theappropriate bioactivity.

Many different types of mutations can give rise to bioactive analogs.For example, conservative changes in the amino acid sequence can beexpected to give rise to analogues that retain one or more bioactivity.It is reasonable to expect that an isolated replacement of a leucinewith an isoleucine or valine, an aspartate with a glutamate, a threoninewith a serine, or a similar replacement of an amino acid with astructurally related amino acid (i.e. conservative mutations) will nothave a major effect on the biological activity of the resultingmolecule. Conservative replacements are those that take place within afamily of amino acids that are related in their side chains. Geneticallyencoded amino acids are can be divided into four families: (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3)nonpolar=alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan; and (4) uncharged polar=glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine,tryptophan, and tyrosine are sometimes classified jointly as aromaticamino acids. In similar fashion, the amino acid repertoire can begrouped as (1) acidic=aspartate, glutamate; (2) basic=lysine, argininehistidine, (3) aliphatic=glycine, alanine, valine, leucine, isoleucine,serine, threonine, with serine and threonine optionally be groupedseparately as aliphatic-hydroxyl;

-   -   (4) aromatic=phenylalanine, tyrosine, tryptophan; (5)        amide=asparagine, glutamine; and (6) sulfur-containing=cysteine        and methionine. (see, for example, Biochemistry, 2nd ed., Ed.        by L. Stryer, WH Freeman and Co.: 1981).

In other embodiments, chemically modified bioactive factors arecontemplated. A polypeptide may be chemically modified to createderivatives by forming covalent or aggregative conjugates with otherchemical moieties, such as glycosyl groups, lipids, phosphate, acetylgroups and the like. Covalent derivatives may be prepared by linking thechemical moieties to functional groups on amino acid side chains or atthe N-terminus or at the C-terminus of the polypeptide. For instance, abioactive factor can be generated which includes a moiety, other thansequences naturally associated with the protein, that binds a componentof the extracellular matrix and enhances localization of the analog tocell surfaces. For example, sequences derived from the fibronectin“type-III repeat”, such as a tetrapeptide sequence R-G-D-S (SEQ ID NO:4) (Pierschbacher et al. (1984) Nature 309:30-3; and Kornblihtt et al.(1985) EMBO 4:1755-9) can be added to a polypeptide factor to supportattachment of the chimeric molecule to a cell through binding ECMcomponents (Ruoslahti et al. (1987) Science 238:491-497; Pierschbacheretal. (1987) J. Biol. Chem. 262:17294-8; Hynes (1987) Cell 48:549-54; andHynes (1992) Cell 69:11-25).

Alternatively, polypeptide growth factors useful in the invention canconsist of active fragments of the factors. The activity of any givenfragment can be readily determined in by methods such as those describedabove. For example, a fragment of bFGF that, when administered accordingto the methods of the invention described herein, is shown to improveperformance in functional tests that is comparable to the performancethat is produced by administration of the full-length bFGF polypeptide,would be an “active fragment” of bFGF. Such active fragments aredescribed, e.g., in Baird and Gage (1997) Proc. Natl. Acad. Sci. U.S.A.,94 (13): 7047-52. It is well within the abilities of skilled artisans todetermine whether a polypeptide growth factor, regardless of size,retains the functional activity of a full length, wild-type polypeptidegrowth factor.

The polypeptide factors useful in the invention are preferablysubstantially purified from their source material, be it cell culture,tissue sample, biological fluid, etc. Substantially purified means thatthe purified material is at least 60% by weight (dry weight) thepolypeptide of interest, e.g., a bFGF polypeptide. Preferably, thepolypeptide composition is at least 75%, more preferably at least 90%,and most preferably at least 99%, by weight, the polypeptide ofinterest. Purity can be measured by any appropriate standard method,e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis. Substantially purified polypeptides can then be combined withother desired components, such as carriers or cells, to give acomposition that is less than 60% composed of polypeptide, so long asthe polypeptide is at sufficient concentration to be effective whenadministered to a patient.

The polypeptide factors useful in the invention can be naturallyoccurring, synthetic, or recombinant molecules consisting of a hybrid orchimeric polypeptide with one portion, for example, being bFGF, and asecond portion being a distinct polypeptide. These factors can bepurified from a biological sample, chemically synthesized, or producedrecombinantly by standard techniques (see.e.g., Ausubel et al., CurrentProtocols in Molecular Biology, New York, John Wiley and Sons, 1993;Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, Suppl.1987).

Although polypeptide growth factors are currently most preferred for usein combination with the cells according to the invention, othertreatment modalities are considered neural stimulants that can becombined with cells according to the invention as well. For example,transcranial magnetic stimulation upregulates monoamines in the brainand is therefore expected to have beneficial effects in conjointadministration with cells.

One group of non-polypeptide neural stimulants that can be used asneural stimulants are neurotransmitter agonists or antagonists. Examplesare antidepressants such as PROZAC, amphetamines, RITALIN, and tricyclicantidepressants such as ELAVIL.

Other useful molecules are differentiation factors such as retinoicacid, which are capable of priming cells to differentiate intofunctioning neurons.

Another class of molecules is the so-called guidance molecules, whichare a class of proteins, normally found in the extracellular matrix,that function to guide cells or cellular processes (axons) to locationsrequired for proper functioning. Examples are the semaphorins, thenetrins, the neuropilins, and the ephrins.

In addition to the above neural stimulants, all of which havewell-established effects on the brain, it is anticipated that otherbioactive compounds that are not considered neural stimulants might beuseful in combination with cells. These alternative compounds aregenerally compounds with well-known effects on other parts of the bodywith more recently discovered effects on cells of the CNS.

One group of alternative compounds includes immunosuppressant moleculesthat are currently used to inhibit rejection of allografts. A preferredclass of such molecules are the immunophilins, such cyclosporin, FK506,and thalidomide. These molecules can exhibit dual action of preventingrejection of the transplanted cells and providing neuroprotectivefunction. Another group of alternative stimulants is the tetracyclines,classically known for their antibiotic effects, but also possessingdesirable neuroprotective effects.

4.4 Cells:

Many different cell types, or mixtures thereof, may be administered to asubject. While not wishing to be limited by theory, it is postulatedthat administered cells may affect the brain in multiple ways. Cells maythemselves become established in the brain and form functionalconnections with neurons. Additionally or alternatively, cells mayproduce factors that stimulate the endogenous nerve cells to form newprocesses and connections. Finally, cells might act to scavenge orotherwise remove or inactivate compounds that inhibit recovery from CNSdamage. In view of these possibilities, it is understood thatessentially any cell possessing one of the above qualities, andparticularly stem cells but potentially even terminally differentiatedcells, might have beneficial effects on brain function. Examples ofterminally differentiated cell types that are known to have beneficialscavenging capabilities are activated lymphocytes and macrophages.

In certain embodiments, the cells of the invention are preferably stemcells that have the capability of giving rise to brain cells in vivo.Particularly preferred cells are multipotential stem cells. Such cellscan be grown in vitro for clinical use. In preferred embodiments, stemcell types that can be used in the invention include neural stem cells,hematopoietic stem cells, embryonic stem cells, teratocarcinoma celllines, and other stem cell types.

The term “stem cell” as used herein refers to cells with the capacityfor unlimited or prolonged self-renewal that can give rise to more thanone type of more differentiated descendant. Preferred stem cells canundergo at least 10 cell divisions (under appropriate conditions) andstill maintain stem cell characteristics. Particularly preferred stemcells can undergo at least 25, 50 or 100 rounds of division withoutlosing stem cell characteristics. With respect to cells, the terms “giverise to” and “produce” are used to mean not just the immediate daughtercells, but all the cells that can eventually trace ancestry to thatcell. “Give rise to” and “produce” also refer to changes in cell typethat might occur without a cell division event. Some differentiatedcells also have the capacity to give rise to cells of greaterdevelopmental potential. Such capacity may be natural under particularcircumstances, or may be induced artificially upon treatment withvarious factors. In either case, the cells may be considered a type ofstem cell for the purposes of the invention. Such stem cells may bereferred to as “induced stem cells” or “differentiated stem cells”.“Processed stem cells” refers to stem cells that have been in any waydisturbed from their natural cellular environment. This includescentrifugation, dissociation, dispersion or other processing. The stemcells contained in an unprocessed tissue sample are not considered“processed stem cells”.

Stem cells are usually rare cell types mixed with other, moredifferentiated cells. For the purposes of the invention, it is possibleto use cell suspensions that comprise only a minority of stem cells.Such an approach is particularly useful with cells derived from a stemcell rich tissue, e.g. bone marrow. In preferred embodiments, stem cellsare enriched such that they are at least 50% pure, meaning that at least50% of the cells are stem cells at the time of administration to asubject. In particularly preferred embodiments, stem cells are at least60%, 70%, 80% or 90% pure.

4.4. 1 General Methods for Stem Cell Culture and Propagation:

Various techniques may be employed to isolate the stem cells of theinvention. Typically, stem cells will be obtained from a tissue sample(e.g. blood, bone marrow, fetal or adult brain tissue, etc.) wherein thedesired stem cells constitute a small percentage of the cells present.In preferred embodiments, the tissue sample is dissociated into a cellsuspension and optionally, various methods are used to enrich for stemcells. Preferred procedures for dissociation of the tissue sample areones that result in as little cell death as possible. For example, stemcells can be dissociated from tissue samples by mechanical means, e.g.,mechanically sheared off with a pipette. In other instances, it will bepossible to dissociate the stem cells from the surrounding tissue byenzymatic digestion. Fluid tissue samples, such as blood, can befractionated by centrifugation and resuspension of certain fractions, ifappropriate. Separation of different cell types and extracellularmaterials may also be achieved by centrifugation or settling in adensity gradient of, for example FICOLL. Stem cell populations may beenriched based on their tendency for continued cell growth as well asspecific cellular markers, e.g., using affinity separation techniques orfluorescence activated cell sorting (FACS).

There are a large number of culture media that exist for culturing cellsfrom animals. Some of these are complex and some are simple. While it isexpected that stem cells may grow in complex media, it will generally bepreferred that the explants be maintained in a simple medium, such asDulbecco's Minimal Essential Media (DMEM), in order to allow moreprecise control over the activation of certain cell populations in atissue sample. The cultures may be maintained in any suitable culturevessel, such as a 12 or 24 well microplate, and may be maintained undertypical culture conditions for cells isolated from the same animal,e.g., such as 37° C. in 5% CO₂. The cultures may be shaken for improvedaeration, the speed of shaking being, for example, 12 rpm.

In general, stem cells can be enriched by detecting and sorting based onidentifying characteristics of the desired cells. For example,monoclonal antibodies are particularly useful for identifying markers(surface membrane proteins, e.g., receptors) associated with particularcell lineages and/or stages of differentiation. Procedures forseparation of the subject progenitor cell may include magneticseparation, using antibody coated magnetic beads, affinitychromatography, and “panning” with antibody attached to a solid matrix,e.g., plate, or other convenient technique. Techniques providingaccurate separation include fluorescence activated cell sorting, whichcan have varying degrees of sophistication, e.g., a plurality of colorchannels, low angle and obtuse light scattering detecting channels,impedance channels, etc.

Antibodies may be conjugated with markers, such as magnetic beads, whichallow for direct separation, biotin, which can be removed with avidin orstreptavidin bound to a support, fluorochromes, which can be used with afluorescence activated cell sorter, or the like, to allow for ease ofseparation of the particular cell type. Any technique may be employedwhich is not unduly detrimental to the viability of the cells.

In addition to using antibodies, it is possible to use other proteinsthat bind to the surface of desired cells. For example, if a desiredcell specifically expresses the EGF receptor, then labeled EGF could beused to detect those cells in much the same way as described for theantibodies above. Certain dyes also stain particular cell populationsand can be used as part of a method for obtaining the desired cells.Stem cells also typically have a distinctive morphology. Stem cellsusually have a large nucleus with a relatively small amount ofcytoplasm.

The selection methods described above may be combined with the use ofselective growth conditions to provide further enrichment. For example,natural and recombinantly engineered cells can be provided as feederlayers to the instant cultures. Such cells can also produce anextracellular matrix that can be used as a substrate for selectionmethods.

It is also possible to contact cell mixtures with an agent that causesproliferation of one or more populations of cells. For instance, amitogen, e.g., a substance that induces mitosis and cell transformationof a particular stem cell type can be used to cause the amplification ofthat population. In this way, cells that are not responsive to theparticular factor tend not to divide while those that are responsivedivide and become a greater proportion of the cell population.

After enrichment it is important to verify that cells obtained have theappropriate characteristics. Cells of the present invention can becharacterized based on responsiveness to growth factors, specific geneexpression, antigenic markers on the surface of such cells, dye stainingand/or basic morphology. It is also valuable to determine the types ofcells that a particular stem cell population can give rise to.

Stem cells can be induced to differentiate into various cell types bychanging the environmental conditions. For example, the subjectprogenitor cells can be recombined with the corresponding embryonictissue to see if the embryonic tissue can instruct the adult cells tocodevelop and codifferentiate. Stem cells can be implanted into one of anumber of regeneration models used in the art, e.g., neural stem cellswill colonize and differentiate in the brain of a rat that has beenlesioned (Gage et al. (1995) Proc. Natl. Acad. Sci. USA, 92:11879-11883; Flax et al. (1998) Nature Biotechnology 16: 1033-1039).Stem cells may be genetically labeled by transfection with a piece offoreign DNA. This labeling allows identification of stem celldescendants from among the host cells. Alternatively, the progenitorcells can be contacted with one or more growth or differentiationfactors which can induce differentiation of the cells. Differentiatedcell types can be identified using the same general methods used toidentify stem cells, e.g. cell surface marker, dye staining etc.

In certain situations it is desirable to measure cell proliferation.Such methods most commonly include determining DNA synthesischaracteristic of cell replication. There are numerous methods in theart for measuring DNA synthesis, any of which may be used according tothe invention. In an embodiment of the invention, DNA synthesis has beendetermined using a radioactive label (3H-thymidine) or labelednucleotide analogues (BrdU) for detection by immunofluorescence.

Growth factors may also be provided in the medium to selectively expandcertain cell populations or to encourage the production ofdifferentiated cell types.

Cells can be sorted by positive and negative selection. For example,positive or negative selection may be achieved by using one or morebiotinylated antibodies, specific for factors on the surface of thetarget cells. The biotinylated antibodies are introduced into the cellculture. After a specified incubation time any biotinylated antibodieswhich have not formed a complex with the target cells are rinsed away.Immobilized avidin matrix is then added to the cell suspension. Theimmobilized avidin matrix binds to the biotinylated antibody/antigencomplex. This suspension can then be centrifuged to separate the avidinmatrix. Alternatively, the avidin may be coupled to magnetic beads suchthat the cells bound to the antibody are magnetically separated fromunbound cells. If the selection is positive, cells bound to the antibodyare resuspended in nutrient medium for continued growth. If theselection is negative, bound cells may be disposed of, while theremaining unbound cells are resuspended for further growth.

Clearly, many other techniques may be utilized for both positive andnegative selection, as long as the desired affinity is provided by theselection element.

Hematopoietic Stem Cells:

Mammalian blood cells provide for an extraordinarily diverse range ofcell types. Three major lineages of blood cells include the lymphoidlineage, e.g. B-cells and T-cells, the myeloid lineage, e.g. monocytes,granulocytes and megakaryocytes, and the erythroid lineage, e.g. redblood cells. Hematopoietic stem cells (HSCs) are cells that can giverise to cells of at least two of the above lineages in addition toproducing daughter cells of equivalent multipotency. In preferredembodiments, the HSCs can give rise to three major blood cell lineages.In addition to giving rise to blood cells, HSCs are capable ofdifferentiating into many other cell types, including brain cells(Eglitis and Mezey (1997) Proc. Natl. Acad. Sci. USA, 94: 4080-4085).

HSCs can be isolated from a variety of tissue types. Bone marrow cellsare a good source of HSCs. Bone marrow cells may be obtained from asource of bone marrow, e.g., iliac crests, tibiae, femora, spine, orother bone cavities. Other sources of human hematopoietic stem cellsinclude embryonic yolk sac, fetal liver, fetal and adult spleen andblood, including adult peripheral blood.

HSCs can be identified both by the types of cells they give rise to andby various cytological markers. HSCs often extrude certain dyes, such asHoechst 33324 and Rhodamine 123 (Bhatia et al. (1998) Nature Med.4:1038). Such dye staining properties can be used to identify HSCs amongother cells of the circulatory system. Antibodies that react withcertain cell markers can also be used to identify and purify HSCs. Forexample, mAb AC133 is thought to specifically bind to HSCs (Miraglia etal. (1997) Blood 90:5013). The Thy-1 molecule is a highly conservedprotein present in the brain and hematopoietic system of rat, mouse andman. The Thy-1 molecule has been identified on rat, mouse and human HSCsand can be useful in identifying HSCs (U.S. Pat. No. 5,914,108). ManyHSCs are CD34+and/or CD38+as well (U.S. Pat. No. 5,840,580). Apopulation of HSCs will often have some variation in cell surfacemarkers and a positive identification may be made on the basis of thepresence of at least two of the above cytological markers.

HSCs can also be distinguished from other more differentiated cell typesby the absence of certain markers. CD3, CD7, CD8, CD10, CD14, CD15,CD19, CD20 and CD33 are all typically absent from HSCs. The absence ofseveral of the above markers adds confidence to the identification ofHSCs. Morphology may also help distinguish an HSC, as described above.

It is understood that HSCs may be identified by an aggregation ofmultiple traits, such as morphology, the presence of certain markers,the absence of other markers, and the types of cells that the putativeHSCs can give rise to. A positive identification does not typicallyrequire detection of all of the above markers.

The culturing of HSCs to give rise to differentiated stem cells can beachieved in many ways. For example, cells may be cultured in a defined,enriched medium such as Iscove's Modified Dulbecco's Medium (IMDM),generally composed of salts, amino acids, vitamins, antibiotics andfetal calf serum. Cultures supplemented with hydrocortisone tend to giverise to myeloid cells, while cultures lacking cortisone tend to giverise to B lymphocytes. To demonstrate that HSCs can develop in cells ofthe erythroid lineage, various conventional methods can be used. Forexample culturing on methylcellulose culture can stimulate formation oferythroid cells. (U.S. Pat. Nos. 5,840,580 and 5,914,108; Metcalf (1977)In: Recent Results in Cancer Research 61. Springer-Verlag Berlin, pp.1-227).

Neural Stem Cells:

Neural stem cells are cells derived from tissue of the adult ordeveloping nervous system that can differentiate into at least one ofthe following fundamental neural lineages: neurons, oligodendroglia andastroglia. Additionally, neural stem cells can also give rise to newNSCs with similar potential. In preferred embodiments, neural stem cellsare multipotential and give rise to cells of most or all of thefundamental neural lineages.

Each of the fundamental neural lineages can be distinguished bydetecting lineage-specific proteins, as well as by morphology. Neuronscan be recognized by detecting, for example, microtubule-associatedprotein 2 (MAP2), tau, certain beta-tubulins (e.g. TuJ1, beta-tubulintype III), certain neurofilament proteins (e.g. neurofilament L or M),neuron-specific enolase, or NeuN. Oligodendrocytes can be recognized bydetecting galactocerebrosidase (GaIC), CNPase, myelin basic protein, or04 protein. Astrocytes can be recognized by the presence of glialfibrillary acid protein (GFAP). Certain NSCs can themselves berecognized by the presence of vimentin or nestin. Typically detection isdone by standard immunostaining techniques using antibodies thatrecognize the desired proteins (Villa et al. (2000) Exp. Neurology 161:67-84). Antibodies for each of the above markers are available from oneor more of the following companies: Chemicon, Sigma-Aldrich,Boehringer-Mannheim, Santa Cruz Biotechnology, Dakopatts AB (Sweden).The expression of genes encoding lineage-specific proteins may also beused to distinguish cells of different lineage. Detection of geneexpression can also be measured by a variety of well-known techniquesincluding in-situ hybridization, fluorescent in-situ hybridization,quantitative rtPCR, Northern blot.

Preferred methods for isolating and propagating NSCs are described inthe following publications: Snyder et al., U.S. Pat. No. 5,958,767;McKay et al., U.S. Pat. No. 5,270,191; Johe, K., U.S. Pat. No.5,753,506; Carpenter, M., U.S. Pat. No. 5,968,829, Weiss et al. U.S.Pat. No. 5,750,376. All of these are herein incorporated by reference.

In general, neural stem cells are maintained in a proliferative,undifferentiated state in the presence of one or more growth factors,for example: bFGF, EGF, TGF-alpha, LIF, or aFGF. Preferred factors arebFGF or EGF. Withdrawal of such factors allows differentiation intocells of distinct lineage. The lineages formed depend on theenvironment. For example, certain NSCs introduced into the brain canform all of the different brain cell types depending on the particularenvironment each cell finds itself in. In culture, the developmentalpathway can be influenced by many factors. For example, CNTF can inducedifferentiation into astrocytes, PDGF can induce formation of neurons,and thyroid hormone (T3) can induce formation of oligodendroglial cells.

In preferred embodiments, neural stem cells are obtained as described inU.S. Pat. No. 5,958,767. This method is described here in brief as anexample of a specific method for preparing NSCs. It is understood thatmany such methods exist and that the details of this method can bemodified to give similar results. In brief, a suspension of primarydissociated neural cells is prepared from the telencephalon of a 15 weekgestational fetus. The suspension is plated on uncoated tissue culturedishes with Dulbecco's Modified Eagle Medium (DMEM) plus F12 medium(1:1) supplemented with N2 medium (Gibco) to which bFGF and heparin orEGF is added. Cell aggregates are dissociated when they grow to a sizelarger than 10 cell diameters in size. Dissociation is performed withtrypsin and the NSC cell suspension is resuspended in growth medium.Dissociated stem cells can be plated on poly-L-lysine coated slides inDMEM+fetal bovine serum to encourage differentiation. Astrocytedifferentiation can be stimulated by co-culturing with primarydissociated cultures of newborn CD-1 mouse brain. Cells may betransfected so as to express a gene that promotes cell division,allowing cell proliferation in vitro without added growth factors.Processes for generating transfected cells are well known in the art. Inpreferred embodiments, the cells are transfected with an amphotrophicreplication-incompetent retroviral vector, and the mitogenic gene isexpressed from the viral LTR region. Preferably, the gene that promotescell division does not encode a neural stimulant. Preferred genes to beexpressed are vmyc, SV-40 T antigen, ras oncogene, polyoma large Tantigen, neu oncogene or combinations thereof. Preferably, suchproliferation-promoting genes and proteins are expressed or active invitro but poorly expressed or inactive in vivo. The vmyc gene appears tobe self-regulating in this manner. Alternatively, inducible promotersthat require a factor, provided in vitro, to stimulate gene expressionmay be used.

Other Stem Cells:

Certain embryonal tumors contain many multipotent cell types. In certainembodiments, cell lines established from these tumors may be used aspart of a method for treating CNS injuries. Useful cell lines derivedfrom embryonal tumors have been described. For example, cells of theNT2/Tera cell line are capable of differentiating into all of the majorneural lineages (U.S. Pat. No. 5,175,103).

Such cells may be isolated from embryonal tumors by any of the generalmethods described above and in U.S. Pat. No. 5,175,103 and in Andrews(1984) Dev. Biol. 103: 285-293. In brief, a human teratocarcinoma cellline (Ntera 2/CI.DI or NT2 cells) can be grown on retinoic acid to forma dense, multi-layered culture. These dense cultures are replated.Small, dense NT2-N cells are loosely associated with an underlying layerof cells. These can be easily dislodged and enriched, yielding a cultureof small, round phase bright cells with some flat contaminating cells.NT2-N cells can be further enriched by culturing with a combination ofmitotic inhibitors, such as cytosine arabinoside. The desired roundcells are resistant to this treatment, while the flat cells do notproliferate. Enrichment of NT2-N cells tending towards a neuraldevelopmental pathway stain with an anti-NF-L antibody (low molecularweight neurofilament protein), while undifferentiated NT2 cells (flatcells) stain with Cam5.2 which reacts with keratins 8 and 18.

Non-cancerous embryonic tissue is also a source for stem cells. Earlyembryonic cells are totipotent, being capable of giving rise to theentire adult organism. As a result, such cells may be cultured to givetotipotent or highly multipotent stem cells. Embryonic stem cells may beused as part of an inventive method for treating CNS injuries. Dependingon culture conditions, these cells may eventually give rise to morecommitted cell types and certain terminally differentiated cell types.Embryonic stem cells may be obtained and cultured as described inThomson et al. (1998) Science 282:1145-1147; Evans et al. (1981) Nature292:154; Martin, G. (1981) Proc. Natl. Acad. Sci. USA 78:7634.

4.5 Administration:

Administration of cells and other treatments may be carried out byvarious methods, and the methods need not be the same for eachcomponent. Generally, when the treatment is a chemical compound, themolecule can be administered by any known route of administration,including intravenously, orally, or intracerebrally (e.g.,intraventricularly, intrathecally, or intracisternally, or directly intothe brain). The dose may vary depending on the method of administration(see Table 2). Doses determined in rats are typically scaled up forhuman treatments. The scaling to be used depends upon the method ofdelivery. If the stimulant is to be delivered systemically (e.g. orallyor intravenously) then the scaling is by body weight, where a typicalrat weighs 300 grams and a typical human weighs 70 kg. If the compoundis to be delivered to the cerebrospinal fluid (e.g. intracisternal,intraventricular), scaling is by brain surface area. A typical rat brainhas a surface area of 1 cm², and a typical human brain has a surfacearea of 1000-10,000 cm², depending upon whether all of the various foldsburied in convolutions are counted or not. If the compound is to bedelivered to the brain tissue, scaling is done by brain mass. A typicalrat has a 2 g brain, while the typical human brain is 2 kg. Thus, if asingle treatment of 0.5 μg given intracisternally is effective in a rat,it would be expected that an intracisternal injection of 0.5 mg would beeffective in a human patient. Of course exact dosages can be adjustedaccording to the weight of the patient and other criteria. It isanticipated that effective dosage for all three general routes ofadministration may range from 0.001-1000 mg total for administration tospinal fluid or brain tissue. In preferred embodiments, the dosage mayrange from 0.01-100 mg, 0.1-10 mg or 0.5-5 mg.

Table 2: Scaling for Dosages of Cells and Stimulants TABLE 2 Scaling fordosages of cells and stimulants Method of Administration Systemic ToSpinal Fluid To Brain Tissue (scale by (scale by (scale by Subject bodyweight) brain surface area) brain weight) Rat 300 grams        1 cm² 2grams Human  70 kg    1000-10,000 cm² 2 kg   

Compounds may be administered in a single dose or they may bedistributed in a series of smaller doses. For example, intracisternaladministration can consist of a single injection given, for example, sixhours after an injury, a pair of injections, given, for example, 24 and48 hours after an injury, or, if necessary, a series of injections of,for example, 0.1 mg/injection, or a 1 mg injection, given biweekly (forexample, every 3-4 days) in a treatment regimen that occurs at least sixhours following the ischemic episode. The treatment regimen may last anumber of weeks.

In certain embodiments, the cells are preferably administered directlyinto the stroke cavity, the spinal fluid, e.g., intraventricularly,intrathecally, or intracisternally. The cells are carried in apharmaceutically acceptable liquid medium, which can contain thebioactive molecule as well. As an alternative, the cells (alone or mixedwith the stimulant) can be administered to the stroke cavity or into thespinal fluid bathing the brain (e.g., intrathecal or intracisternaladministration). Cells may also be injected into the region of the brainsurrounding the area(s) of damage, and cells may be given systemically,given the ability of certain stem cells to migrate to the appropriateposition in the brain. If the cells are to be injected into the strokecavity, the ventricles of the brain, or into the brain tissue, thepatient's head is immobilized in a standard stereotactic frame, and thesite of administration of the cells is located by standard CT or MRIscan. A small-bore hole is drilled in the skull, and the cells areinjected into the desired location using a syringe. Cells are scaledaccording to method of administration as detailed in table 2. Generally,between 10⁶ and 10¹² cells are administered in total, preferably between10⁷ and 10¹¹ and more preferably between 10⁸ and 10¹⁰. Multiple celladministrations can be used, generally at least 2-7 days apart.

Administration of cells and treatments will be preferably carried outanywhere from several hours or several days following the injury toseveral weeks or even months following the stroke. In preferredembodiments, administration is carried out at least 6, 10, 12 or 24hours after the injury has occurred. It is anticipated that exactdosages for both cells and neural stimulants may be adjusted by themedical practitioner in response to the particular needs andcharacteristics of the patient. In general it is expected that theoptimal dosage is high enough to be effective but low enough to avoidprovoking excessive inflammatory response, which can becounter-productive. By determining the level of inflammatory response,one could determine whether a particular dosage rate is too high to giveoptimal effectiveness. The methods of administration presented hereinare preferred because they permit precise control and modulation of doselevels and because the area to which cells and stimulants are appliedcan be carefully controlled. In preferred embodiments, the neuralstimulant is not produced from a transgene contained within one or moreof the administered cells.

Other desirable compounds may be administered with the cells and neuralstimulants. For example, immunosuppressants and antibiotics are usefulfor preventing graft rejection and infection, respectively. Furthermore,as discussed above, these types of compounds may have additionalbeneficial effects.

Common methods of administering the cells and bioactive factors of thepresent invention to subjects, particularly human subjects, which aredescribed in detail herein, include injection or implantation of thecells and/or neural stimulants into target sites in the subjects. Thecells and factors of the invention can be inserted into a deliverydevice which facilitates introduction by injection or implantation intothe subjects. Such delivery devices include tubes, e.g., catheters, forinjecting cells and fluids into the body of a recipient subject. In apreferred embodiment, the tubes additionally have a needle, e.g., asyringe, through which the cells of the invention can be introduced intothe subject at a desired location. The cells and factors of theinvention can be inserted into such a delivery device, e.g., a syringe,in different forms. For example, the cells or factors can be suspendedin a solution or embedded in a support matrix when contained in such adelivery device. As used herein, the term “solution” includes apharmaceutically acceptable carrier or diluent in which the cells of theinvention remain viable. Pharmaceutically acceptable carriers anddiluents include saline, aqueous buffer solutions, solvents and/ordispersion media. The use of such carriers and diluents is well known inthe art. The solution is preferably sterile and fluid. Preferably, thesolution is stable under the conditions of manufacture and storage andpreserved against the contaminating action of microorganisms such asbacteria and fungi through the use of, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.Solutions of the invention can be prepared by incorporating progenitorcells as described herein in a pharmaceutically acceptable carrier ordiluent and, as required, other ingredients enumerated above, followedby filtered sterilization. Optionally, cells may be administered onsupport matrices. Support matrices in which cells can be incorporated orembedded include matrices which are recipient-compatible and whichdegrade into products which are not harmful to the recipient. Naturaland/or synthetic biodegradable matrices are examples of such matrices.Natural biodegradable matrices include plasma clots, e.g., derived froma mammal, and collagen matrices. Synthetic biodegradable matricesinclude synthetic polymers such as polyanhydrides, polyorthoesters, andpolylactic acid. Other examples of synthetic polymers and methods ofincorporating or embedding cells into these matrices are known in theart. See e.g., U.S. Pat. No. 4,298,002 and U.S. Pat. No. 5,308,701.These matrices provide support and protection for the cells in vivo.

Cells and neural stimulants of the invention may be administeredtogether in a pharmaceutical composition. Appropriate compositions mayinclude all compositions usually employed for systemically or locallyadministering drugs. The pharmaceutically acceptable carrier should besubstantially inert, so as not to act with the active components orinterfere with cell viability. Suitable inert carriers include water,alcohol polyethylene glycol, propylene glycol and the like.

To prepare the pharmaceutical compositions of this invention, aneffective amount of the particular neural stimulant and cells as activeingredients are combined with a pharmaceutically acceptable carrier,which carrier may take a wide variety of forms depending on the form ofpreparation desired for administration. These pharmaceuticalcompositions are desirable in unitary dosage form suitable,particularly, for administration percutaneously, or by parenteralinjection. Any of the usual pharmaceutical media may be employed suchas, for example, water, glycols, oils, alcohols and the like in the caseof oral liquid preparations such as suspensions, syrups, elixirs andsolutions; or solid carriers such as starches, sugars, kaolin,lubricants, binders, disintegrating agents and the like in the case ofpowders, pills, capsules, and tablets. For parenteral compositions, thecarrier will usually comprise sterile water, at least in large part,though other ingredients, for example, to aid solubility and cellviability, may be included. Other ingredients may include antioxidants,viscosity stabilizers, chelating agents, buffers, preservatives. Ifdesired, further ingredients may be incorporated in the compositions,e.g. anti-inflammatory agents, antibacterials, antifungals,disinfectants, vitamins, antibiotics.

Examples of antioxidants comprise butylated hydroxytoluene, butylatedhydroxyanisole, propyl gallate, citric acid and ethoxyquin; examples ofchelating agents include disodium edetate and ethanehydroxy diphosphate;examples of buffers comprise citric acid, sodium citrate, boric acid,borax, and disodium hydrogen phosphate; and examples of preservativesare methyl parahydroxybenzoate, ethyl parahydroxybenzoate, dehydroaceticacid, salicylic acid and benzoic acid. Injectable solutions, forexample, may be prepared in which the carrier comprises saline solution,glucose solution or a mixture of saline and glucose solution. Injectablesuspensions may also be prepared in which case appropriate liquidcarriers, suspending agents and the like may be employed. Also includedare solid form preparations which are intended to be converted, shortlybefore use, to liquid form preparations. In the compositions suitablefor percutaneous administration, the carrier optionally comprises apenetration enhancing agent and/or a suitable wetting agent, optionallycombined with suitable additives of any nature in minor proportions,which additives do not introduce a significant deleterious effect on theskin.

It is especially advantageous to formulate the subject compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used in the specification and claims herein refersto physically discrete units suitable as unitary dosages, each unitcontaining a predetermined quantity of active ingredient calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. Examples of such dosage unit forms are capsules,injectable solutions or suspensions, teaspoonfuls, tablespoonfuls andthe like, and segregated multiples thereof.

Particular compositions for use in the method of the present inventionare those wherein the neural stimulant is formulated inliposome-containing compositions. Liposomes are artificial vesiclesformed by amphiphatic molecules such as polar lipids, for example,phosphatidyl cholines, ethanolamines and serines, sphingomyelins,cardiolipins, plasmalogens, phosphatidic acids and cerebrosides.Liposomes are formed when suitable amphipathic molecules are allowed toswell in water or aqueous solutions to form liquid crystals usually ofmultilayer structure comprised of many bilayers separated from eachother by aqueous material (also referred to as coarse liposomes).Another type of liposome known to be consisting of a single bilayerencapsulating aqueous material is referred to as a unilamellar vesicle.If water-soluble materials are included in the aqueous phase during theswelling of the lipids they become entrapped in the aqueous layerbetween the lipid bilayers.

Water-soluble active ingredients are encapsulated in the aqueous spacesbetween the molecular layers. A lipid soluble active ingredient of aneural stimulant, such as an organic mimetic, is predominantlyincorporated into the lipid layers, although polar head groups mayprotrude from the layer into the aqueous space. The encapsulation ofthese compounds can be achieved by a number of methods. The method mostcommonly used involves casting a thin film of phospholipid onto thewalls of a flask by evaporation from an organic solvent. When this filmis dispersed in a suitable aqueous medium, multilamellar liposomes areformed. Upon suitable sonication, the coarse liposomes form smallersimilarly closed vesicles.

Water-soluble active ingredients are usually incorporated by dispersingthe cast film with an aqueous solution of the compound. Theunencapsulated compound is then removed by centrifugation,chromatography, dialysis or other art-known suitable procedures. Thelipid-soluble active ingredient is usually incorporated by dissolving itin the organic solvent with the phospholipid prior to casting the film.If the solubility of the material in the lipid phase is not exceeded orthe amount present is not in excess of that which can be bound to thelipid, liposomes prepared by the above method usually contain most ofthe material bound in the lipid bilayers; separation of the liposomesfrom unencapsulated material is not required.

A particularly convenient method for preparing liposome formulated formsof neural stimulants is the method described in EP-A-253,619,incorporated herein by reference. In this method, single bilayeredliposomes containing encapsulated active ingredients are prepared bydissolving the lipid component in an organic medium, injecting theorganic solution of the lipid component under pressure into an aqueouscomponent while simultaneously mixing the organic and aqueous componentswith a high speed homogenizer or mixing means, whereupon the liposomesare formed spontaneously.

The single bilayered liposomes containing the encapsulated neuralstimulant can be mixed with cells and then employed directly or they canbe employed in a suitable pharmaceutically acceptable carrier forlocalized administration. The viscosity of the liposomes can beincreased by the addition of one or more suitable thickening agents suchas, for example xanthan gum, hydroxypropyl cellulose, hydroxypropylmethylcellulose and mixtures thereof. The aqueous component may consistof water alone or it may contain electrolytes, buffered systems andother ingredients, such as, for example, preservatives. Suitableelectrolytes which can be employed include metal salts such as alkalimetal and alkaline earth metal salts. The preferred metal salts arecalcium chloride, sodium chloride and potassium chloride. Theconcentration of the electrolyte may vary from zero to 260 mM,preferably from 5 mM to 160 mM. The aqueous component is placed in asuitable vessel which can be adapted to effect homogenization byeffecting great turbulence during the injection of the organiccomponent. Homogenization of the two components can be accomplishedwithin the vessel, or, alternatively, the aqueous and organic componentsmay be injected separately into a mixing means which is located outsidethe vessel. In the latter case, the liposomes are formed in the mixingmeans and then transferred to another vessel for collection purpose.

The organic component consists of a suitable non-toxic, pharmaceuticallyacceptable solvent such as, for example ethanol, glycerol, propyleneglycol and polyethylene glycol, and a suitable phospholipid which issoluble in the solvent. Suitable phospholipids which can be employedinclude lecithin, phosphatidylcholine, phosphatidylserine,phosphatidylethanol-amine, phosphatidylinositol, lysophosphatidylcholineand phosphatidyl glycerol, for example. Other lipophilic additives maybe employed in order to selectively modify the characteristics of theliposomes. Examples of such other additives include stearylamine,phosphatidic acid, tocopherol, cholesterol and lanolin extracts.

In addition, other ingredients which can prevent oxidation of thephospholipids may be added to the organic component. Examples of suchother ingredients include tocopherol, butylated hydroxyanisole,butylated hydroxytoluene, ascorbyl palmitate and ascorbyl oleate.Preservatives such a benzoic acid, methyl paraben and propyl paraben mayalso be added.

Methods of introduction may also be provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinacious biopharmaceuticals. A variety ofbiocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of a bioactive factor at a particulartarget site.

An essential feature of certain embodiments of the implant can be thelinear release of the therapeutic, which can be achieved through themanipulation of the polymer composition and form. By choice of monomercomposition or polymerization technique, the amount of water, porosityand consequent permeability characteristics can be controlled. Theselection of the shape, size, polymer, and method for implantation canbe determined on an individual basis according to the disorder to betreated and the individual patient response. The generation of suchimplants is generally known in the art. See, for example, ConciseEncylopedia of Medical & Dental Materials, ed. by David Williams (MITPress: Cambridge, Mass., 1990); and the Sabel et al. U.S. Pat. No.4,883,666.

In another embodiment of an implant cells are encapsulated inimplantable hollow fibers or the like. Such fibers can be pre-spun andsubsequently loaded with the cell source (Aebischer et al. U.S. Pat. No.4,892,538; Aebischer et al. U.S. Pat. No. 5,106,627; Hoffman et al.(1990) Expt. Neurobiol. 110:39-44; Jaeger et al. (1990) Prog. Brain Res.82:41-46; and Aebischer et al. (1991) J. Biomech. Eng. 113:178-183), orcan be co-extruded with a polymer which acts to form a polymeric coatabout the cells (Lim U.S. Pat. No. 4,391,909; Sefton U.S. Pat. No.4,353,888; Sugamori et al. (1989) Trans. Am. Artif. Intern. Organs35:791-799; Sefton et al. (1987) Biotehnol. Bioeng. 29:1135-1143; andAebischer et al. (1991) Biomaterials 12:50-55). Such encapsulated cellscan then be combined with a neural stimulant.

It is anticipated that, for convenience, it would be desirable forneural stimulants and cells to be packaged together into kits. Kits mayinclude dose-size-specific ampules or aliquots of cells and/or neuralstimulants. Kits may also contain devices to be used in administeringthe components of the conjoint administration. Such devices have beendescribed above. In certain embodiments, wherein the cells are to beobtained from the patient, cultured, and readministered to the patient,the kit may comprise a device for obtaining a cell sample from thepatient from which stem cells will be cultured.

In certain aspects, practitioners of the present invention may employ,unless otherwise indicated, conventional techniques of cell biology,cell culture, molecular biology, transgenic biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are described in the literature. See, for example,Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes 1 and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

5. EXAMPLES Example 1 Intracisternal Neural Stem Cells (NSC) and GrowthFactors Enhance Stoke Recovery

In this example, fetal mouse neural stem cells (NSC) with or withoutbasic fibroblast growth factor (bFGF) were administered intracisternallyin a model of stroke recovery in rats. Male Sprague-Dawley rats, 300-350grams, were handled for one week before surgery. They received anantibiotic, cefazolin sodium (40 mg/kg, i.p.), one day before strokesurgery. On the day of stroke surgery, animals were anesthetized by 2%halothane in a nitric oxide/oxygen mixture (2:1). Focal cerebralinfarction, (stroke) was performed by proximal electrocoagulation of themiddle cerebral artery, as described previously (Kawamata et al. (1999)Exp. Neurol. 158, 89-96; Tamura et al. (1981) J. Cereb. Blood FlowMetab. 1, 53-60). Specifically, the artery was occluded from justproximal to the olfactory tract to the inferior cerebral vein, withoutremoving the zygomatic arch or transsection of the facial nerve. Thistechnique produces a robust and reproducible infarct, or region of celldeath, in the dorsolateral cerebral cortex and underlying striatum.Animals received another injection of cefazolin sodium (40 mg/kg, i.p.)immediately after surgery. They were then allowed to awaken fromanesthesia.

Twenty-four hours after stroke surgery, animals received anintracisternal injection of either: (1) vehicle, (2) NSC (10⁶ cells),(3) bFGF (0.5 μg), or (4) NSC+bFGF. Intracisternal injection in 50 μltotal volume was done through percutaneous injection into the cisternamagna under halothane anesthesia. This same procedure was repeated twodays later so that animals received treatment on days 1 and 3 followingstroke. Cyclosporin, an immunosuppressant, was administered at 10 mg/kg,i.p. for the duration of the experiment.

The cerebral infarcts produced by the procedure cause sensorimotordysfunction of the contralateral hindlimb and forelimb. For the nextmonth following stroke, a number of neurological tests were done toassess sensorimotor function of the contralateral limbs. These testsinclude both the forelimb and hindlimb placing tests which test theanimal's ability to place the limb on a tabletop in response to visual,tactile, proprioceptive, and whisker stimulation. In addition, a bodyswing test was done that measures the side to side preferences of theanimal as he is held suspended by his tail above a tabletop. Finally,the spontaneous limb use test is done which measures the animal'spropensity to use each forelimb spontaneously as he rears up to explorethe inside of a narrow glass cylinder. The forelimb and hindlimb placingtest, as well as the spontaneous limb use test reflect both cortical andstriatal function. The body swing test is mainly a measure of striatalfunction.

The results of these tests are shown in FIGS. 1A-1D. Panels (A) and (B)show placing activity of the affected forelimb and hindlimb(contralateral to the side of the stroke in the brain). Panel (C) showsthe body swing test, and panel (D) shows the spontaneous limb use test.In each instance, normal behavior is indicated by the data obtained onthe day before surgery (−1 day). In each case, animals showed markedlyabnormal behavior on the day following surgery. There was then a slowspontaneous recovery that was incomplete. FIGS. 1A-1D show that on thelimb placing tests all three treatments: NSC, bFGF and the combination,significantly enhanced recovery compared to placebo. There was a similartrend in the spontaneous limb use test. No differences among treatmentscompared to placebo were seen on the body swing test. In addition,although this was nonsignificant, a trend toward superior enhancement offunction was seen in the combination group compared to the NSC and bFGFgroups alone.

At one month following stroke, animals were sacrificed, brains wereremoved and sectioned and stained with H & E. Infarct volume wasdetermined via image analysis, as described previously (Kawamata et al.(1996) J. Cereb. Blood Flow Metab. 16, 542-547; Kawamata et al. (1997)Proc. Nat. Acad. Sci. 94, 8179-8184). No significant differences wereseen in infarct volume among groups, although there was a trend towardslightly smaller infarct volume in the groups receiving NSC. The stemcells that were transplanted contain the lacZ reporter gene and express#-galactosidase. X-gal histochemistry was done to examine the locationof these cells post-transplant. Indeed, the cells had migrated fromtheir site of installation in the cisterna magna to positionssurrounding the focal stroke in the right hemisphere.

In summary, this experiment showed that NSC and/or bFGF administeredintracisternally starting one day after stroke can significantly enhancesensorimotor recovery of the contralateral limbs. This improvement waslargely confined to tests reflecting cortical function. No significantdifferences were seen in infarct volume among the groups, suggestingthat NSC and bFGF produced recovery-promoting effects through othermechanisms than the prevention of cell death. These mechanisms mayinclude establishing new connections in undamaged parts of brain.Moreover in this first experiment, the combination of NSC and bFGFappeared to be slightly superior to either treatment alone.

Example 2 Direct Intracerebral Administration of NSC and IntracisternalAdministration of bFGF Enhance Recovery in Rat Stroke Model

In a second experiment, NSC were injected directly into the brain intotissue surrounding focal strokes. bFGF was injected intracisternally, asbefore. In this experiment only one administration of NSC or bFGF wasperformed at one day after stroke. Under these conditions, we clearlyobserved the superiority of NSC+bFGF compared to either treatment alone.

In this experiment, animals were handled for one week before surgery. Inaddition, they were trained on an additional test, the paw reaching test(see below) for 10 days before surgery. As before, they receivedcefazolin sodium (40 mg/kg, i.p.) before surgery. On the day of surgery,electrocoagulation of the proximal middle cerebral artery was done, asdescribed previously (Kawamata et al. (1996) J. Cereb. Blood Flow Metab.16, 542-547; Kawamata et al. (1997) Proc. Nat. Acad. Sci. 94, 8179-8184;Kawamata et al. (1999) Exp. Neurol. 158, 89-96). They received anotherinjection of cefazolin sodium, 40 mg/kg, i.p. after surgery.

At one day after stroke, animals received either: (1) vehicle injectioninto periinfarct tissue, and vehicle injection into the cisterna magna,(2) NSC (10⁶ cells) into periinfarct tissue and vehicle into thecisterna magna, (3) vehicle into periinfarct tissue and bFGF (0.5 μg)into the cisterna magna or (4) the combination NSC (10⁶ cells) intoperiinfarct tissue and bFGF (0.5 μg) into the cisterna magna.

These injections were done with a volume of 25 μl each under 2%halothane anesthesia. NSC was injected into striatal tissue at themargins of focal infarcts. bFGF was injected percutaneously into thecisterna magna (intracisternal injection) as described previously(Kawamata et al. (1996) J. Cereb. Blood Flow Metab. 16, 542-547;Kawamata et al. (1997) Proc. Nat. Acad. Sci. 94, 8179-8184; Kawamata etal. (1999) Exp. Neurol. 158, 89-96). Rats also received cyclosporin, animmunosuppressant (10 mg/kg, i.p. per day), throughout the duration ofthe experiment.

As before, a number of behavioral tests were done for the next monthfollowing stroke. These tests included the forelimb and hindlimb placingtests, the body swing test, and the spontaneous limb use test, asdescribed in Example 1. In addition, another test was done, the pawreaching test. Animals were trained on this test before stroke surgery,and then were tested once at the end of the experiment. This testexamines the animal's ability to reach through the bars of his cage tograb and eat food pellets with the impaired (contralateral) forepaw.Normally, animals have about 100% accuracy in performing this task.Following stroke, it drops down to about 10%.

The results of these behavioral tests are shown in FIGS. 2A-2D and 3.Again, all three treated groups: NSC, bFGF, and the combination ofNSC+bFGF, showed superiority in recovery on the forelimb and hindlimbplacing tests compared to placebo. Again, there was a trend towards bestrecovery in the combination group. In the body swing test, NSC treatmentalone did not show advantage over placebo, but both the bFGF andcombination groups did. In the spontaneous limb use test, only thecombination group showed a trend toward improved outcome. Finally, inthe paw reaching test, the combination group appeared to showsuperiority compared to either treatment alone. Histological evaluationof these brains is still pending.

In summary, in this experiment NSC was injected directly into tissuebordering focal strokes. bFGF was administered intracisternally. Each ofthese treatments, when delivered alone, improved behavioral outcome onsome tests. For each test, the combination treatment appeared to bebetter than either treatment alone. This was particularly apparent onthe spontaneous limb use and paw reaching tests. This experimentsupports the notion that the combination of stem cell and growth factortreatment is superior to either treatment alone in enhancing strokerecovery. Both of the examples above were done using only one dose ofNSC and growth factor. Further studies are underway to define the doseresponse characteristics of this interaction.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of treating a subject with CNS ischemic damage, the methodcomprising administering to the subject: neural stem cells; and afibroblast growth factor (FGF); wherein the conjoint administration ofthe neural stem cells and the FGF ameliorates effects of CNS ischemicdamage.
 2. A method as set forth in claim 1, wherein the FGF is a basicfibroblast growth factor.
 3. A method as set forth in claim 1, whereinthe neural stem cells are obtained from fetal blood.
 4. A method as setforth in claim 1, wherein the neural stem cells are administeredintravenously, intracerebrally, intraventricularly or intracisternally.5. A method as set forth in claim 1, wherein the neural stem cells andthe FGF are both administered intravenously.
 6. A method of treating asubject with brain damage resulting from stroke, the method comprisingadministering to the subject: neural stem cells; and a fibroblast growthfactor (FGF); wherein the conjoint treatment with the neural stem cellsand the FGF ameliorates effects of brain damage.
 7. A method as setforth in claim 6, wherein the conjoint treatment is initiated at least 6hours after the stroke was diagnosed.
 8. A method as set forth in claim6, wherein the FGF is a basic fibroblast growth factor.
 9. A method asset forth in claim 6, wherein the neural stem cells are obtained fromfetal blood.
 10. A method as set forth in claim 6, wherein the neuralstem cells are administered intravenously, intracerebrally,intraventricularly or intracisternally.
 11. A method as set forth inclaim 6, wherein the neural stem cells and the FGF are both administeredintravenously.
 12. A method of treating a subject with CNS ischemicdamage, the method comprising administering to the subject: stem cellsobtained from blood; and a fibroblast growth factor (FGF); wherein theconjoint administration of the stem cells obtained from blood and theFGF ameliorates effects of CNS ischemic damage.
 13. A method as setforth in claim 12, wherein the FGF is a basic fibroblast growth factor.14. A method as set forth in claim 12, wherein the neural stem cells areobtained from fetal blood.
 15. A method as set forth in claim 12,wherein the neural stem cells are administered intravenously,intracerebrally, intraventricularly or intracisternally.
 16. A method asset forth in claim 12, wherein the neural stem cells and the FGF areboth administered intravenously.
 17. A method of treating a subject withCNS ischemic damage, the method comprising administering to the subject:fetal blood cells; and a fibroblast growth factor (FGF); wherein theconjoint administration of the fetal blood cells and the FGF ameliorateseffects of CNS ischemic damage.
 18. A method as set forth in claim 17,wherein the FGF is a basic fibroblast growth factor.
 19. A method as setforth in claim 17, wherein the neural stem cells are administeredintravenously, intracerebrally, intraventricularly or intracisternally.20. A method as set forth in claim 17, wherein the neural stem cells andthe FGF are both administered intravenously.